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Journal of Bacteriology, February 1999, p. 1364-1368, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Virulence Plasmid of Salmonella typhimurium Is Self-Transmissible

Brian M. M. Ahmer,* Mimi Tran, and Fred Heffron

Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201-3098

Received 13 October 1998/Accepted 2 December 1998


    ABSTRACT
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Most isolates of Salmonella enterica serovar Typhimurium contain a 90-kb virulence plasmid. This plasmid is reported to be mobilizable but nonconjugative. However, we have determined that the virulence plasmid of strains LT2, 14028, and SR-11 is indeed self-transmissible. The plasmid of strain SL1344 is not. Optimal conjugation frequency requires filter matings on M9 minimal glucose plates with a recipient strain lacking the virulence plasmid. These conditions result in a frequency of 2.9 × 10-4 transconjugants/donor. Matings on Luria-Bertani plates, liquid matings, or matings with a recipient strain carrying the virulence plasmid reduce the efficiency by up to 400-fold. Homologs of the F plasmid conjugation genes are physically located on the virulence plasmid and are required for the conjugative phenotype.


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Salmonella enterica serovar Typhimurium (commonly referred to as Salmonella typhimurium) has been studied extensively, both as a model of fundamental genetic mechanisms and as a model of host-pathogen interactions. This bacterium is a broad-host-range pathogen that causes gastroenteritis in humans and cattle and typhoid-like disease in mice. Most S. typhimurium isolates (88%) carry a plasmid of 60 MDa (roughly 90 kb) (15). This plasmid has been given a variety of names, including pSLT, MP10, pRQ28, pSTV, the cryptic plasmid, and the virulence plasmid (7, 15, 25, 28, 30, 34). We will refer to this plasmid simply as the virulence plasmid of a particular host strain.

The most apparent consequence of virulence plasmid carriage is to enhance the growth rate of the bacterium during the systemic phase of disease (13). This phenotype is conferred by an 8-kb region of the plasmid that encodes the spv (Salmonella plasmid virulence) genes (11, 12). The product of the spv regulatory locus, SpvR, in combination with the alternative sigma factor RpoS, is required for the expression of the four structural genes spvABCD (35 and references therein). The molecular functions of SpvA, SpvB, SpvC, and SpvD have not been determined, although it appears that SpvD is secreted (8). Other loci on the plasmid include the pef (plasmid-encoded fimbriae) region, which has been implicated in bacterial adhesion to intestinal epithelial cells, and the srg region which includes rck (resistance to complement killing), a homolog of dsbA (disulfide bond isomerase), and a homolog of the AraC family of transcriptional regulators (1, 3, 10, 14). Another interesting gene on the virulence plasmid is tlpA, which encodes an apparent thermometer in that it regulates its own transcription according to temperature (17). The downstream targets of this regulator have not yet been identified.

Isolation of MudJ mutations on the virulence plasmid. We recently performed a genetic screen to identify genes regulated by SirA, a transcriptional regulator of genes within SPI1 (Salmonella pathogenicity island 1) (2, 18). This screen resulted in the isolation of 74 transcriptional fusions (MudJ transposon insertions) that respond to sirA expressed from a multicopy plasmid. However, only 10 of the 74 fusions were found to respond to sirA in its natural chromosomal context. These 10 fusions were defined as class 1 and are definitively regulated by sirA. The class 1 fusions are located within SPI1, SPI4, and SPI5 (2). The remaining 64 fusions are probably not regulated directly by SirA but are expressed as an indirect effect of sirA overexpression. These 64 fusions were defined as class 2. By cloning and sequencing the genomic DNA flanking the class 2 insertions we discovered that many of the fusions are located on the virulence plasmid (Table 1). The known plasmid genes disrupted include pefC, tlpA, and spv.

                              
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  		TABLE 1.   Strains used in this study and their abilities to act as virulence plasmid conjugative donors

The virulence plasmid contains genes homologous to the conjugation genes of F and R-100 plasmids. We were able to sequence the genomic DNA surrounding 53 of the 64 MudJ mutations described above. Database searches indicated that 15 of the 53 mutations are within homologs of the F and R-100 plasmid conjugation genes (Table 1). Consistent with this, Boyd and Hartl reported that 50% of Salmonella isolates contain an F plasmid (5). More recently these authors determined that there is a correlation in Salmonella isolates between the presence of several virulence plasmid loci and the F plasmid locus, traD (6). Based on this correlation, they hypothesized that the F plasmid and the virulence plasmid might be one and the same.

The F plasmid homologs could be located on the virulence plasmid, the chromosome, or on a second plasmid in the cell. To determine their physical location, pulsed-field gel electrophoresis was used to separate the virulence plasmid from the rest of the Salmonella genome, and Southern blotting was used to identify the MudJ insertions (20). Strains carrying MudJ mutations within known virulence plasmid loci were used as positive controls (spv, tlpA, and pefC). Negative controls were a plasmidless strain (BA770), a wild-type strain that does not contain a MudJ mutation (14028), and a strain that contains a MudJ mutation in the chromosome (hilA::MudJ). Ethidium bromide staining of the pulsed-field gel showed that the virulence plasmid was present in all strains except the plasmidless strain, BA770 (Fig. 1A). The plasmids of the wild type and the hilA mutant are slightly smaller than the others, consistent with the lack of a MudJ insertion. The identity of this plasmid was confirmed by using Southern hybridization with the kanamycin resistance gene of MudJ (Fig. 1B). The control strains that contain MudJ insertions in known virulence plasmid loci yield positive hybridization signals (pefC::MudJ, spv::MudJ, and tlpA::MudJ). The negative control strains do not (wild type, plasmidless, and hilA::MudJ). The virulence plasmid of strains containing MudJ insertions within F plasmid homologs (trbC::MudJ, traP::MudJ, traD::MudJ, and trbI::MudJ) also yields positive hybridization signals. This conclusively demonstrates that the F plasmid homologs are physically located on the virulence plasmid. It also suggests that the F plasmid detected by Boyd and Hartl in 50% of Salmonella isolates is actually the virulence plasmid of these strains (5).


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  		FIG. 1.   Homologs of the F plasmid conjugation genes are physically located on the 90-kb virulence plasmid of S. typhimurium. DNA was prepared according to the methods described in reference 36. Pulsed-field gel electrophoresis utilized a 1% 0.5× Tris-borate-EDTA agarose gel with separation by a Bio-Rad CHEF DRII at 200 V. The separation was run for 5 h with a switching time that ramped from 6 to 150 s, 5 h at 6 to 12 s, and 10 h at 6 to 36 s. (A) The gel was stained with ethidium bromide. (B) Detection of the kanamycin resistance gene of MudJ by Southern hybridization after transfer to a positively charged nylon membrane obtained from Boehringer Mannheim. A random prime DNA labeling kit (Boehringer Mannheim) and [32P]dCTP were used to label the 1.2-kb SmaI fragment from pUC4K-KIXX (Pharmacia), which contains the kanamycin resistance gene.

The conjugation genes of the virulence plasmid are functional. Previous reports have stated that the virulence plasmid of S. typhimurium is mobilizable but not self-transmissible (19, 26-29). However, these reports did not focus on the transmissibility of the virulence plasmid. This fact, coupled with the large number of conjugation genes located on the virulence plasmid, suggested that the virulence plasmid might be self-transmissible under certain conditions. To test this hypothesis, we assayed the ability of several strains to act as virulence plasmid donors in conjugation experiments (Table 1). In each case, the donor bacteria contained a MudJ insertion on the virulence plasmid. This served two purposes. First, recipients that had acquired the virulence plasmid could be selected by using the kanamycin resistance of the MudJ insertion. Second, using donors that had MudJ mutations in various genes of the virulence plasmid allowed us to determine which plasmid genes are required for conjugation. The recipient strain was BA770, which is resistant to nalidixic acid (allowing counterselection) and has been cured of the virulence plasmid (3). All donors containing a MudJ insertion in a conjugation gene homolog (traC, traD, traG, traW, traP, orfG, trbC, or trbI) failed to conjugate, either due to the direct disruption of the gene itself or polarity on downstream genes. Donors containing a MudJ insertion elsewhere on the virulence plasmid (spv, pefC, and tlpA) were conjugative according to a qualitative assay (all yielded conjugation frequencies of approximately 10-4 transconjugants/donor; Table 1).

A quantitative analysis of conjugation frequency under optimal conditions yielded a frequency of 2.9 × 10-4 transconjugants/donor (optimal conditions utilized filter matings on M9 minimal glucose plates with a recipient strain lacking the virulence plasmid; Table 2). This conjugation frequency is much lower than that of the Escherichia coli F plasmid but is reasonable considering that most natural self-transmissible plasmids yield frequencies of between 10-3 and 10-5 (23). The F plasmid of E. coli K-12 conjugates with an unusually high frequency of 0.2 to 1.0, because a repressor of the conjugation genes, finO, has been disrupted by an IS3 element (9). It is also possible that currently undiscovered conditions could result in higher virulence plasmid conjugation frequencies.

                              
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  		TABLE 2.   Conjugation frequencies of the S. typhimurium virulence plasmid under various conditions

Because the donor and recipient strains in these experiments were isogenic, it is possible that the transconjugants obtained were actually donors that had spontaneously acquired resistance to nalidixic acid. To eliminate this possibility, two types of control experiment were performed. The first control was to perform mock matings in the absence of a recipient strain. These experiments determined the frequency of spontaneous nalidixic acid resistance by the donors. These mock matings were performed exactly like actual matings, with three separate cultures on three separate days. Many of the mock matings resulted in no spontaneous nalidixic acid-resistant mutants, so an average frequency was not calculated (the detection limit for mutation frequency was 10-9). Instead, the highest frequency observed, during growth on either Luria-Bertani (LB) plates or M9 glucose, was reported (Table 2; 9.0 × 10-8 on LB and 1.1 × 10-7 on M9). This highest frequency of spontaneous nalidixic acid resistance is much lower than the observed conjugation frequencies (Table 2). Therefore, spontaneous drug resistance was not a factor in these experiments.

The second experiment was to physically observe the transferred virulence plasmid in the recipient strain. This requires the recipient to have a genome that is easily distinguished from that of the donor. Therefore, E. coli was chosen to be the recipient in matings with Salmonella. Filter matings were performed on M9 minimal glucose plates with the donor strain BA1541 (spv::MudJ) and recipient strain BA769 (E. coli W3110 Nalr). Transconjugants were obtained at a frequency of approximately 2 × 10-5 transconjugants/donor. Genomic DNA was isolated from the transconjugants, digested with XbaI, and examined by pulsed-field gel electrophoresis (Fig. 2A). Ethidium bromide staining of the pulsed-field gel shows that the restriction pattern of the transconjugant genomes matches that of the E. coli recipient, except for the acquisition of the virulence plasmid from the Salmonella donor. The identity of the virulence plasmid was confirmed by subsequent Southern hybridization with the kanamycin resistance gene of MudJ (Fig. 2B). Because the restriction patterns of the E. coli and Salmonella genomes are different, it is easily seen that the three transconjugants are E. coli bacteria that have obtained the virulence plasmid, rather than donors that have become spontaneously resistant to nalidixic acid. This is clear evidence that conjugation has occurred.


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  		FIG. 2.   Visualization of virulence plasmid transfer from Salmonella to E. coli. (A) Pulsed-field gel stained with ethidium bromide. (B) Southern hybridization of the pulsed-field gel with a probe specific to the kanamycin resistance gene of MudJ. The virulence plasmid is present in the donor Salmonella strain, BA1541 (spv::MudJ), and the three transconjugants but is not present in the recipient E. coli strain, BA769. Pulsed-field gel electrophoresis and Southern blotting were performed as described in the legend to Fig. 1.

Factors affecting conjugation frequency. Several factors were found to alter the conjugation frequency of the S. typhimurium virulence plasmid. First, the conjugation frequency is 70-fold lower when filter matings are performed on LB plates rather than on M9 minimal glucose plates (Table 2). LB plates contain a rich mixture of yeast components that can be used as biosynthetic precursors and as a carbon and energy source. M9 minimal glucose plates, on the other hand, contain only glucose as a carbon source and contain no biosynthetic precursors. This suggests that conjugation proficiency is regulated in response to environmental conditions. This could involve gene expression changes in the donor, the recipient, or both.

Second, liquid matings result in fewer transconjugants per donor than filter matings with M9 medium (Table 2). However, unlike the situation with filter matings, in which the use of M9 medium results in 70-fold higher efficiencies than the use of LB medium, liquid matings in either medium result in roughly the same frequency. This frequency is intermediate to those obtained with the filter matings on M9 glucose and LB plates (Table 2).

Third, the presence of a virulence plasmid in the recipient strain inhibits conjugation approximately 400-fold (Table 2; recipient strain IR715). This is probably due to a surface exclusion process mediated by the plasmid in the recipient strain. This phenomenon is common among plasmids utilizing an F plasmid-like conjugative apparatus (9). The traS and traT genes of the F plasmid are responsible for surface exclusion, and it is known that the S. typhimurium virulence plasmid encodes a homolog of traT (24, 32, 33).

The F plasmid homologs were originally isolated by screening for transcriptional fusions that respond to sirA carried on a multicopy plasmid (2). Because SirA regulates genes required for enteropathogenesis in the bovine model, we hypothesized that SirA may also activate conjugation functions in the intestine. This inherently makes sense, given the large numbers of potential mating partners present in the intestinal environment. Therefore, the effect of a sirA mutation on conjugation frequency was tested. We found a small, but statistically significant, decrease in conjugation efficiency for a sirA mutant donor on either LB or M9 minimal glucose plates (two- to threefold decrease; P = 0.003 on LB plates, P = 0.0096 on M9 glucose; Table 2). However, this is a very small effect, compared to the effects of varying the medium composition. Also, the SirA-regulated MudJ fusions in the virulence plasmid transfer region are found in both the sense and antisense orientations. Although the transfer region of the F plasmid is known to incorporate antisense transcripts (9), we conclude that the regulatory effect of sirA on these genes is probably indirect and not of significant consequence.

Strain background does not explain the previous literature. Because the virulence plasmid has been reported to be nonconjugative, we wanted to determine why our results differed from those of previous studies (19, 26-29). One hypothesis was that there was a difference in strain background. We were utilizing a virulent isolate of S. typhimurium (14028), while historically Salmonella geneticists have used strain LT2. To test this hypothesis we compared the conjugation frequencies of virulence plasmids from several S. typhimurium strains (14028, LT2, SR-11, and SL1344). P22 phage transduction was used to move the spv::MudJ mutation from BA1541 (14028 spv::MudJ) into LT2, SL1344, and SR-11 (strain constructions and lineages are described in Table 1). The spv::MudJ mutation was chosen because it is located directly opposite the conjugation genes on the plasmid (4). Because the virulence plasmid is roughly 90 kb and P22 can package only 45 kb, this should prevent the cotransduction of the MudJ marker and the functional conjugation genes present on the plasmid of 14028. Three transductants from each strain construction were tested for conjugation proficiency. Surprisingly, all three spv::MudJ transductants of strains SR-11 and LT2 (BA901a,b,c and BA902a,b,c; Table 1) acted as virulence plasmid donors at a frequency similar to that of 14028, but all three isolates of strain SL1344 were nonconjugative (BA903a,b,c; Table 1). On a later occasion, all of these strains were constructed again, and the conjugation experiments were repeated. The result was the same: the virulence plasmids of strains LT2 and SR-11 are self-transmissible, but that of SL1344 is not.

Although it appears that strain background cannot explain the discrepancy between our results and those of previous studies, there are other potential explanations. One possibility is that previous investigators utilized conditions that are not optimal for virulence plasmid conjugation. Because conjugation was not the focus of these reports, all of the methods attempted were not listed. However, commonly utilized conditions for conjugation include liquid matings and filter matings on LB plates. Since we determined that conjugation frequency of the virulence plasmid is reduced by using either of these conditions, this is a likely explanation. It is also likely that the recipient strain in previous experiments contained a virulence plasmid. This greatly decreases the conjugation frequency. In addition, some investigators have tested the transmissibility of plasmids marked with Tn10 (19, 27). However, the location of Tn10 on the virulence plasmid was unknown. If the transposon disrupted the conjugation genes, this would also explain the lack of conjugation. In any case, there are several factors that could contribute to the previous lack of detected conjugation.

The demonstration that the virulence plasmid of S. typhimurium is self-transmissible provides an example of horizontal gene transfer. The fact that this genetic exchange involves virulence factors has implications for the evolution of enteric pathogens such as Salmonella. The horizontal transfer of genetic material by conjugation is likely to increase the evolutionary rate at which pathogens can test new virulence gene combinations.


    ACKNOWLEDGMENTS

This work was supported by grant AI22933 from the United States National Institutes of Health.

We thank Cindy Arvidson for a critical reading of the manuscript.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201-3098. Phone: (503) 494-6841. Fax: (503) 494-6862. E-mail: ahmerb{at}ohsu.edu.


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Journal of Bacteriology, February 1999, p. 1364-1368, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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